DESCRIPTION (adapted from the application) Recent data suggest that Acyl CoA binding protein (ACBP) has equally important roles in lipid metabolism. ACBP is a ubiquitous protein that interacts with long-chain fatty acyl-CoA. In vitro subcellular studies suggest that ACBP may affect intracellular enzymes involved in long-chain fatty acyl-CoA synthesis, Beta-oxidation, phospholipid synthesis, and fatty acid synthesis. In contrast, almost nothing is known of ACBP function in lipid metabolism of intact whole mammalian cells. Mouse embryonic stem (ES) cells represent an undifferentiated normal cell type derived from the inner cell mass of a blastocyst. ES cells can be maintained in culture without loss of totipotency, and can be reintroduced into a host embryo. A major objective of this proposal is to produce ACBP overexpressing and gene disrupted / ablated mouse ES cell lines to determine how changes in ACBP expression affect subcellular localization and long-chain fatty acyl-CoA metabolism in intact cells. Furthermore, ES cells can be induced to differentiate into a fibroblast cell type by retinoic acid induction, thereby providing an additional model of differentiating effects of ACBP on fatty acid metabolism. The specific aims for the proposed studies are: 1) Produce mouse recombinant ACBP and select endpoint assays for the assessment of ACBP function in vitro. 2) Overexpress/gene disrupt ACBP in mouse ES cells, and determine how specific intracellular localizations are affected by changes in ACBP. 3) Determine how changes in ACBP expression affect lipid metabolism in intact mouse ES cells. During the first phase of the program, Dr. McArthur will determine the effect of overexpression of ACBP on mouse ES cell function and ACBP localization. After completing his Ph.D. degree, Dr. McArthur will then isolate the mouse ACBP gene, construct a targeting vector for disruption of one and both ACBP alleles, and produce single and double ACBP gene disrupted mouse ES cell lines. He will assess at the cellular level the effect of ACBP gene disruption on fatty acid metabolism. Not only will the proposed work provide important information about the function of ACBP in mammalian cells, it will allow Dr. McArthur the opportunity to develop his scientific career as an independent investigator.